Multiple pathological conditions including muscle trauma, surgical damage, and myopathies can lead to volumetric muscle loss (VML) resulting in unrecoverable muscle function. To date, the treatments for VML including the muscle flap or graft are not always effective and are hindered by limited tissue availability and donor site morbidity. The creation of engineered muscle tissue with functional myotendinous junctions, tendon- bone anchors and innervation will not only restore the function of a complex tissue, e.g. a small facial muscle following traumatic injury or disease, but can also be used as a model for studying developmental muscle biology and muscle pharmacology. To address the need for skeletal muscle replacement tissue, our laboratory has developed a tissue-engineered functional skeletal muscle unit (SMU) of appropriate size and function for clinical use in situations of small VML injuries, such as those found in the hand and face [1-8]. Our SMU is a multi-phasic composite tissue consisting of engineered muscle tissue with engineered tendon and/or bone- tendon anchor ends. After twenty-eight days of exposure in vivo to a VML environment, our constructs exhibited significant alteration to the SMU phenotype including uniaxial aligned muscle fibers encased in an extensive extra-cellular matrix, complete bone healing at the bone attachment, formation of enthesis and myotendinous interfaces , extensive vascularization and innervation with concomitant formation of neuromuscular junctions (8). This exciting technology actually results in the addition of new muscle fibers to the repair area. Our findings show that our multi-phasic composite tissue survives implantation, survives the mechanical loads placed on them in vivo, and develops the necessary interfaces needed to advance the phenotype toward adult muscle structure and function. While our technology shows great promise for the repair of damaged muscle, a lot of work remains to determine how best to utilize this technology to obtain optimal recovery of muscle function to an injury site. For example, we need more complete recovery of native muscle forces. An additional concern regarding the use of this novel and innovative technology in humans is the safety of use of stem/precursor cell-derived tissue in patients. It is imperative that the primary or stromal cells used for the generation of these tissue constructs pose no acute or long-term threat after implantation. The main concern in the field is the ability to ascertain that no undifferentiated cels persist in the transplanted construct that might later lead to aberrant cellular behavior such as cancer. Thus, to progress this technology towards clinical application, the mechanisms of graft tissue integration, regeneration, and repair must be elucidated. The overall goal of this proposal is to use VML as a model for looking mechanistically at the integration and regeneration of our SMU tissue following implantation into an appropriate inductive tissue environment. This work has broad implications for stem cell biology, musculoskeletal biology and regenerative medicine as well as direct clinical relevance for the treatment of musculoskeletal tissue injuries.